Ec-CdtC Dictates Resistance to EGA and Alters Intracellular Trafficking of Ec-CdtB.

<p>(A) CHO-A745 cells were intoxicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143977#pone.0143977.g001" target="_blank">Fig 1</a> except that all wells were additionally treated with 12.5 μM EGA. (B) CHO-A745 cells were seeded at 8 x 10³ cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with 1μM Ec-CDT holotoxin or 1 μM Ec-CdtAB for 4 or 16 h. Phosphorylated H₂AX (anti-pH₂AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH₂AX induced by Ec-CDT holotoxin was set at 100% and used to normalize signal from CdtAB for each time point. Graphs represent average values from three independent experiments, each performed at least 3 times. *p value = 0.0121 calculated by unpaired two-tailed t test (Prism 5, GraphPad). (C, D) CHO-A745 cells were seeded at 2 x 10⁴ cells/well on 8-well chambered slides and allowed to adhere overnight. The next day, cells were incubated on ice with 100 μM Ec-CDT holotoxin, Ec-CdtAB or Ec-CdtBC for 30 min, washed and incubated at 37°C for 60 minutes. Cells were then fixed, stained, and imaged as described in Methods [anti-Ec-CdtB (green) and EEA1 or Rab9 antibody (red)]. White scale bars at the left panel of each treatment indicate 10 μm and the right insert panel indicate 2 μm. Quantification of microscopy results was performed using Pearson's coefficient values indicating colocalization of the Ec-CdtB signal with the EEA1 or Rab9 enriched vesicles. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.